INFECTION AND IMMUNITY, July 2005, p. 4423–4426
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 7
Cross-Recognition of N-Formylmethionine Peptides Is a General
Characteristic of H2-M3-Restricted CD8?T Cells
Alexander Ploss,1,2An Tran,1Ewa Menet,1Ingrid Leiner,1and Eric G. Pamer1*
Infectious Diseases Service, Department of Medicine and Laboratory of Antimicrobial Immunity, Immunology
Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue,
New York, New York 10021,1and Immunology Program, Weill Graduate School of Medical Sciences of Cornell
University, New York, New York 100212
Received 12 January 2005/Returned for modification 1 February 2005/Accepted 17 February 2005
H2-M3-restricted CD8?T cells can exhibit cross-reactivity to different bacterially derived N-formylmethi-
onine peptides. The extent of this promiscuity is unclear. We deleted the nonredundant fMIVTLF epitope and
found that Listeria monocytogenes still primed fMIVTLF-specific T cells. Thus, cross-reactivity appears to be a
more general characteristic of H2-M3-restricted T cells.
Infection with Listeria monocytogenes induces antigen-
specific CD8?T cells that clear infection. H2-M3 major
histocompatibility complex (MHC) class Ib molecules have
a defined role in antibacterial immunity and present at least
three L. monocytogenes-derived, N-formylmethionine pep-
tides (fMIGWII, fMIVTLF, and fMIVIL) to CD8?T cells
(6, 9, 14). Recently, it was demonstrated that many H2-M3-
restricted CD8?T cells specific for the dominant fMIGWII
epitope cross-react with other N-formyl bacterial peptides, a
feature that is unusual for the adaptive immune system (5, 13).
To test whether epitope cross-reactivity is unique to fMIGWII-
specific T cells or is shared by other H2-M3-restricted CD8?T
cells, we generated a mutant strain of L. monocytogenes that
lacks the subdominant fMIVTLF epitope (L. monocytogenes
fMIVTLFneg) (Fig. 1A). The fMIVTLF sequence is nonredun-
dant; i.e., it is not contained within any other predicted protein
sequences in the L. monocytogenes proteome (tblastn search
[www.ncbi.nlm.nih.gov/BLAST]). To generate the L. monocy-
togenes fMIVTLFnegstrain, the attM region of L. monocyto-
genes was mutated in the fMIVTLF epitope to fMIVIL, which
has also been defined as another H2-M3-restricted epitope (6)
as described previously (13). A 510-bp fragment of the attM
region was amplified from genomic DNA from L. monocyto-
genes using the following primers: 5?attM (Invitrogen, Carls-
bad, CA) (CCGGAATTCCCGTGGGTCATTAAGAAAATAG)
and 3?attM (AAAACTGCAGCCCATGTTTTAGCAATTGG
TCG). The fragment was cloned into the pBluescript II SK
vector (Stratagene, La Jolla, CA), and the fMIVTLF sequence
was mutated by in vitro mutagenesis using the following prim-
ers: fMIVTLFmutF (TTTTTAATGATTGTAATATTAATTT
ATTCAGCGTATTCC) and fMIVTLFmutR (GGTATACGC
TGAATAAATTAATATTACAATCATTAAAAA). The attM
fragment containing the mutated sequence was cloned into the
pkSV7 vector, and the mutation was then incorporated into the
chromosome of Listeria monocytogenes 10403s by homologous
recombination as described previously (16). To confirm the pres-
ence of the mutation, the attM gene region was amplified from
the genomic DNA preparation of the L. monocytogenes wild type
(wt) and L. monocytogenes fMIVTLFnegstrain by PCR and sub-
sequent sequencing (Memorial Sloan-Kettering Cancer Center
sequencing core facility). The kinetics of growth of L. monocyto-
genes fMIVTLFnegand wild-type L. monocytogenes 10403s are
indistinguishable (Fig. 1B). The epitope mutation did not impair
virulence, as measured by bacterial counts of the spleens and
livers of infected mice 72 h following infection.
To test whether deletion of fMIVTLF abrogates T-cell prim-
ing, we adoptively transferred 5,6-carboxyfluorescein diacetate
succinimidyl ester (CFSE)-labeled C10.4 (H2-M3:fMIVTLF)
(2) or L9.6 (H2-Kd:p60217-225-specific) (8) T-cell receptor
(TCR) transgenic (tg) CD8?T cells into congenic recipients
and measured expansion of transferred cells 4 days following
infection with a sublethal dose of wild-type L. monocytogenes
or epitope-deleted strains (Fig. 1D). It is noteworthy that
C10.4-specific T cells do not recognize fMIVIL (data not
shown). Whereas wild-type L. monocytogenes primes both the
C10.4 and L9.6 TCR tg T cells, infection with L. monocytogenes
218S, which lacks the p60217-225epitope (15), does not lead to
proliferation of H2-Kd-restricted CD8?T cells. In contrast,
priming of C10.4 TCR tg T cells is not affected by the deletion
of the fMIVTLF epitope. These results demonstrate that de-
letion of this H2-M3-restricted epitope does not prevent prim-
ing of T cells with specificity for the deleted epitope, extending
the finding that H2-M3-restricted CD8?T cells are promiscu-
ous to a second epitope.
To determine whether early activation and the expansion
kinetics of T cells responding to fMIVTLF or non-fMIVTLF
epitopes are similar, we analyzed adoptively transferred C10.4
TCR tg T cells following infection with wild-type L. monocy-
togenes or the fMIVTLFnegstrain (Fig. 2). H2-M3:fMIVTLF-
specific T cells were primed efficiently in the absence of their
cognate epitope, up-regulating CD25, CD44, and CD69 ex-
pression and down-regulating CD62L expression (Fig. 2A) and
proliferating with kinetics identical to that of T cells isolated
from mice infected with wild-type bacteria (Fig. 2B). Cross-
reactive ligand recognition following infection with L. mono-
* Corresponding author. Mailing address: Infectious Diseases Ser-
vice, Department of Medicine and Laboratory of Antimicrobial Im-
munity, Immunology Program, Sloan-Kettering Institute, Memorial
Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY
10021. Phone: (212) 639-7809. Fax: (212) 717-3021. E-mail: pamere
cytogenes fMIVTLFnegresults in endogenous fMIVTLF-spe-
cific T-cell populations of similar sizes, as determined by
tetramer staining 6 days following primary infection (Fig. 3A).
H2-M3 tetramers were generated as described previously (7).
These results indicate that priming, in the absence or presence
of fMIVTLF, leads to a similar activation.
To test whether T cells primed by non-fMIVTLF epitopes
are fully functional, we analyzed the cytokine profile of H2-
M3-restricted T cells after primary infection with wild-type or
fMIVTLFnegL. monocytogenes (Fig. 3B and C). Restimulation
of splenocytes 6 days after infection with fMIGWII peptide
served as an internal control and induced the release of gamma
interferon (Fig. 3B) and tumor necrosis factor alpha (Fig. 3C)
from similar numbers of CD8?T cells. Interestingly, compa-
rable numbers of cytokine-producing CD8?T cells were also
detected when the T cells were restimulated with fMIVTLF
peptide, irrespective of whether the cells were primed by
To determine whether in vivo cytolytic activity against
fMIVTLF is generated by immunization with L. monocytogenes
fMIVTLFneg, we assessed epitope-specific cytolytic activity in
mice infected with wild-type L. monocytogenes or L. monocy-
togenes fMIVTLFneg(Fig. 3D) (3, 12). Splenocytes from
C57BL/6 mice were labeled with high and low concentrations
of CFSE. Target cells labeled with a high concentration of
CFSE (CFSEhigh) were coated with fMIVTLF peptide. Equal
numbers of the two populations were adoptively transferred
into syngeneic recipients that had been infected 5 days earlier
with a sublethal dose of wild-type L. monocytogenes or L.
monocytogenes fMIVTLFnegor left uninfected. In vivo cytolytic
activity was measured by disappearance of the fMIVTLF-
pulsed CFSEhightarget cells 18 h after transfer and was readily
detectable in mice infected with wt or fMIVTLFnegL. mono-
cytogenes. The degree of in vivo cytolysis of fMIVTLF-bearing
target cells was comparable in mice infected with either strain
(average decrease of wt versus fMIVTLFnegstrain, 70% versus
75%). These data show that H2-M3-restricted T cells primed
in the absence of fMIVTLF are fully functional and exhibit
effector functions in vivo in response to fMIVTLF.
In this study we demonstrate that deletion of the nonredun-
dant fMIVTLF epitope from L. monocytogenes does not impair
priming or differentiation of “fMIVTLF-specific” T cells. An-
tigen cross-reactivity has been reported for T cells specific for
several MHC class Ib molecules. Qa-1-restricted CD8?T cells
recognize an epitope derived from the GroEL molecule of
Salmonella enterica serovar Typhimurium and expand in re-
sponse to a peptide derived from self heat shock protein Hsp60
(11). Similarly, CD1-restricted CD8?T cells recognize glyco-
lipids from endogenous and bacterial sources (17). Our study
provides further evidence that cross-reactive ligand recogni-
tion is a feature common to many H2-M3-restricted T cells.
How can promiscuous antigen recognition be explained?
One of the characteristics of nonclassical MHC class Ib mol-
ecules, such as CD1 or H2-M3, is their lack of polymorphism
(10). This class of molecules has evolved to present relatively
invariant antigens, in the case of H2-M3, N-formylated, hydro-
phobic peptides. As a consequence of the structural features of
the H2-M3 binding groove, only a few endogenous mitochon-
drially derived peptides and some bacterial peptides fulfill the
requirements for this molecular shape. The surface created by
complexes of different ligands bound to H2-M3 and recognized
by T cells may be very similar; thus, distinct MHC peptide
complexes may not be readily distinguished by T cells.
How can H2-M3-restricted CD8?T cells that recognize
multiple ligands escape the selection processes in the thy-
mus? A diverse population of self peptides was shown to be
essential for the in vivo development of CD4 T cells. This
requirement for peptide diversity indicates that the interac-
tion between self peptides and T-cell receptors during pos-
itive selection is highly specific (1). The number of endog-
enous H2-M3-restricted ligands that could allow for
selection of T cells is very limited. Mitochondria are the only
source for N-formylmethionine peptides in eukaryotic cells
but encode only 13 peptides of which only a few actually
contribute to positive selection (4). Hence, the specificity of
T cells that become positively selected is highly skewed. The
limited number of endogenous M3 ligands might also affect
negative selection, which might account for the unusually
FIG. 1. Generation and characterization of the L. monocytogenes
fMIVTLFnegstrain. (A) The MIVTLF sequence in the attM region of
L. monocytogenes 10403s was mutated to MIVIL to generate an L.
monocytogenes strain lacking the fMIVTLF sequence (L. monocyto-
genes fMIVTLFneg). (B) L. monocytogenes (L.m.) fMIVTLFneg(open
circles) grows with kinetics similar to that of L. monocytogenes 10403s
(black squares). Cultures were inoculated with 1 ml of cultures grown
overnight in 1,000 ml brain heart infusion broth. Cultures were grown
at 37°C, and the bacterial concentration was determined at the indi-
cated time points. (C) Bacterial numbers in spleens and livers of
C57BL/6 mice 72 h after primary infection (infected with 5,000 bacte-
ria) with L. monocytogenes fMIVTLFnegor L. monocytogenes 10403s.
The values are shown as means ? standard errors (error bars) of three
mice per group. (D) Priming of H2-M3- but not H2-Kd-restricted
CD8?T cells in the absence of the cognate peptide antigen. Flow
cytometric histograms are gated on donor CD8?Thy1.1?T cells and
are representative of three mice per group.
high degree of cross-reactivity of H2-M3-restricted CD8?T
cells in the periphery. Many of the vast number of endoge-
nous ligands, which negatively select MHC class Ia-re-
stricted CD8?T cells may resemble pathogen-derived se-
quences. On the other hand, H2-M3-restricted TCRs, which
are negatively selected on a few ligands, may result in a
broader TCR repertoire with a greater predilection for pep-
FIG. 2. Kinetics of fMIVTLF-specific CD8?T-cell activation early after primary infection. (A) CFSE-labeled splenocytes (1 ? 106) from C10.4
B6.PL TCR tg mice were adoptively transferred into C57BL/6 recipients. The mice were left untreated (noninfected [non-inf.]) (white bars) or were
infected with 5,000 L. monocytogenes (L.m.) 10403s (black bars) or L. monocytogenes fMIVTLFneg(hatched bars). At the indicated time points,
splenocytes were stained with anti-CD8, anti-Thy1.1, and one of the indicated antibodies (CD25, CD44, CD62L, and CD69). The surface
expression levels for the various activation markers of the transferred C10.4 TCR tg T cells (CD8?Thy1.1?) are plotted as the mean fluorescent
intensity (MFI). (B) T-cell proliferation was measured at the indicated time point postinfection (post inf.) by analysis of CFSE dilution. Flow
cytometric histograms are gated on donor CD8?Thy1.1?T cells and are representative of three mice per group. The number in each plot
represents the percentage of C10.4 TCR tg T cells that underwent at least one round of division.
VOL. 73, 2005NOTES4425
We thank Maggie Zhong for excellent technical support and mem-
bers of the lab for insightful discussions.
This work was supported by National Institutes of Health grant
AI49602 and by a Cancer Research Institute predoctoral fellowship
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Editor: F. C. Fang
FIG. 3. H2-M3-restricted CD8?T-cell expansion following L.
monocytogenes fMIVTLFneginfection. (A) Six days following infec-
tion with L. monocytogenes (L.m.) 10403s or L. monocytogenes
fMIVTLFneg, the total number of fMIGWII- or fMIVTLF:H2-M3-
tetramer-positive CD8?cells was determined. (B and C) The total
number of CD8?T cells producing gamma interferon (IFN-?)
(B) or tumor necrosis factor alpha (TNF-?) (C) 6 days following
infection with L. monocytogenes 10403s or L. monocytogenes
fMIVTLFnegwere determined by ex vivo intracellular cytokine
staining. non-inf., noninfected. (D) fMIVTLF-specific in vivo cytol-
ysis is induced by immunization with L. monocytogenes fMIVTLFneg
infection. The numbers above the graphs refer to individual mice
used in one experiment. The experiment was repeated twice.